Phytophthora spp. - rising threat for the natural and agro ecosystems in Bulgaria

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1 162 Bulgarian Journal of Agricultural Science, 20 (No 1) 2014, Agricultural Academy Phytophthora spp. - rising threat for the natural and agro ecosystems in Bulgaria AgroBioInstitute, bg Sofia, Bulgaria Abstract Lyubenova, A. B. and S. B. Slavov, Phytophthora spp. - rising threat for the natural and agro ecosystems in Bulgaria. Bulg. J. Agric. Sci., 20: Species from genus Phytophthora are known as pathogens mainly on agricultural plants in Bulgaria, causing diseases as potato blight disease, black shank on tobacco, blights on peppers and tomatoes, etc. They also cause problems in orchards and nurseries. Less data concerning these plant pathogens are available for forestry and in fact there was no comprehensive investigation of the Phytophthora biodiversity in Bulgarian ecosystems. The problem with the increasing number of incidents of severe disease outbreaks caused by introduced Phytophthora pathogens in different types of ecosystems is discussed in this review. Some of the Phytophthora species known as inhabitants in the European countries, but still not found in Bulgaria, are pointed and discussed as a potential invaders for the country and increased danger for the natural and agro ecosystems in the country. The awareness is focused on some factors that might influence this process, like human activity, global climate changes and natural interspesific hybridization. Key words: Phytophthora, biodiversity, distribution, introduction Introduction Literally translated from Greek the name Phytophthora means plant destroyer. Phytophthora is a genus of plant pathogens that completely correspond to its name, comprising approximately 100 species described in the literature by now (Kroon et al., 2012). The number of the newly described species is growing rapidly, in response of the increased interest among scientists to this group of devastating plant pathogens. The best studied and probably the most popular species of all the members of this genus is Phytophthora infestans, the causing agent of the potato late blight. When first appeared on potato crop in Ireland in 1845 this new pathogen have completely destroyed the potato yield in two subsequent years, resulting in mass starvation, migration and the death of millions of people in Ireland at that time (Erwin and Ribeiro, 1996). P. infestans is the first microorganism proven to be responsible for disease, and the work on it initiated the science of plant pathology (Judelson and Blanco, 2005). Beside its importance as a crop plant pathogens Phytophthora spp. appears to be involved in oak decline phenomenon, which is of great concern in Europe since 1980 s and also is associated with other prominent cases of trees mortality within natural forest ecosystems (Jung et al., 2002; Brasier and Jung, 2004; Jung et al., 2005; Oszako, 2007). In this review we focus on the problem with the increasing number of incidents of severe disease outbreaks caused by introduced Phytophthora pathogens in different types of ecosystems. Particular concern is caused, when such cases lead to permanent disturbing of the natural balance in intact ecosystems. It is ongoing process, the consequences of which are hard to predict. We discuss some of the factors that might influence this process, like human activity, global changes in climate and natural interspesific hybridization. Taxonomy Microorganisms from the genus Phytophthora have been considered to be typical fungi for a very long time. Traditionally, Phytophthora have been included in Oomycetes, within Fungi, based on the superficial resemblance of the hyphae and apparent similarities in the sexual reproduction (Dick, 2001). anetta7@abv.bg

2 Phytophthora spp. - Rising Threat for the Natural and Agro Ecosystems in Bulgaria 163 Based on the evolutionary phylogeny it is clear now that Oomycetes are not fungi, and are closely related to diatoms and golden-brown algae (Lamour et al., 2007). Despite that, in broad sense species from genus Phytophthora are still considered as fungi, or fungus-like organisms, mainly due to the similarities in the growing pattern and ecological behavior. According to the classification of Dick (2001), the genus Phytophthora belongs to Kingdom Straminipila, class Peronosporomycetes (Oomycetes), a diverse group of microorganisms, including saprotrophs, facultative and obligate parasites on plants, animals and crustaceans. Economically the most important members of the Peronosporomycetes are the phytoparasites, which include the obligate downy mildews (Peronospora spp., Bremia spp., Plasmopara spp., Pseudoperonospora spp, Sclerospora spp., etc.), white rusts (Albugo spp.), semi-biotrophs from Phytophthora spp. and the saprotriophs from genus Pythium. In the revision of the classification of the Eukaryotes (Adl et al., 2005) which incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies, Phytophthora spp. fall in the super-group Chromalveolata. Distantly, the Fungi belong to the super-group Opisthokonta, together with Animalia. The chromalveolates brings together the stramenopiles (oomycetes, diatoms, and brown algae) and the alveolates (apicomplexans, ciliates and dinoflagellates). Genome sequences of Phytophthora ramorum and Phytophthora sojae reveals similarity to photosynthetic organisms such as red algae or cyanobacteria. These and other analyses support the view that a photosynthetic ancestor founded the stramenopiles. In other words: It appears that devastating pathogens, such as Phytophthora, have evolved from benign phototrophic ancestors (Lamour et al., 2007). In addition, oomycetes appears to be more closely related to the apicomplexans, including parasites such as Plasmodium and Toxoplasma, than to Fungi. Oomycetes and apicomplexans share similar mechanisms of pathogenesis, and that might be a reflection of their common evolutionary history (Lamour et al., 2007). Life cycle of Phytophthora in relation with the pathogenesis of the disease The thallus of Phytophthora, as well as the most oomycetes is filamentous, composed of hyphae, and is diploid in its vegetative stage (Erwin and Ribeiro, 1996; Judelson and Blanco, 2005). Hyphae are developed in the convergent evolutionary process, and are analogous to hyphae of the Eumycota (Dick, 2001). Oomycetes has several clearly defined developmental stages in their life cycle (Walker and Van West, 2007). Several types of spores have important role in the life and the disease cycles of Phytophthora species. In nature, the survival of Phytophthora spp. as a saprophyte is limited, and spores are required to allow movement to new host. The asexual spore, called sporangium is multinucleate spore, which is capable to convert its cytoplasm into multiple spores (zoospores) (Judelson and Blanco, 2005). The process of zoosporogenesis is considered one of the fastest developmental processes in any biological system (Walker and Van West, 2007). The zoospore is the primary infecting unit and its role is the transmission of the pathogen from host to host. It is a free-swimming spore, possessing two flagella of different length. Once released, they can swim for hours. Directional swimming of zoospores towards chemical, nutrient or electrical gradients are typical of zoospore taxis (Walker and Van West, 2007). After the motile period zoospores encyst, involving the loss of both flagella and the formation of a primary cell wall (Judelson and Blanco, 2005). The resulting cyst germinates by producing germ tubes and mycelia. After direct germination, hyphae ramify through living plant tissue, forming feeding relationship. The epidemic spread of the diseases, caused Phytophthora spp. to be often based on the rapid dispersal from host to host by zoospores that are released from sporangia. Some of the species have sporangia, which are able to detach from the sporangiophores and when shed, are both airborn and water-born (Erwin and Ribeiro, 1996). The motile zoospores extend the range of Phytophthora beyond the landing cites of air-born sporangia. The sporangia are metabolically active, but vulnerable to death, caused by the harsh environmental conditions as solar radiation and drought. By contrast more true fungi produce desiccated spores which can remain dormant for long periods of time, but which can consequently miss opportunities for host infection ( Judelson and Blanco, 2005). The increase of inoculums of Phytophthora spp. is caused by the rapid production of sporangia and zoospores from infected plant tissues, when the environmental conditions, the most important of which is the presence of free water are favorable. The evolutionary strategies of straminipilious fungi and eumycote fungi are similar in that they depend on large population with ephemeral generation times (Dick, 2001). All of the oomycetous microorganisms have short generation time and great reproductive capacity. For this reason, caused by Phytophthora spp. diseases are generally considered to be multicyclic (Erwin and Ribeiro, 1996). Some of the Phytophthora species forms chlamydospores. These asexual spores have thicker walls and are capable of persisting in soil and plant tissue for long periods. They are of particular importance as survival propagules to some species (Erwin and Ribeiro, 1996).

3 164 The sexual spores (оospores) are the most persistent of all propagules produced by Phytophthora. As a product of sexual reproduction, the resulted progeny have been considered more variable than the parent cultures, and might be a source of new variants with increased virulence. The heterothallic species produce oospores mainly when the A1 and A2 (opposite) mating type grow together. Species that produce oospores in single cultures are designated homothallic (Erwin and Ribeiro, 1996). The function of mating of the A1 and A2 types has been considered to be a true hybridization phenomenon, in which factors that condition parasitism and many other characters would recombine in the progeny. This is supported of the results of tests with nuclear genetic markers, showing that recombination of factors occurs (Erwin and Ribeiro, 1996). Alien and native Phytophthora spp. The introduction and spread of exotic and invasive species is one of the most important problems in conservation biology. Defining invasive species, not only the causing agents of emerging infective plant diseases should be included as invaders, but also fungi with unknown impact (saprotrophs and mycorrhizal fungi) and fungi with economic, but unknown ecological impact (pathogens of crop plants). It has been estimated that % of plant pathogens worldwide are alien in the locations where they were recorded. Some of the most devastating alien species belong to the genus Phytophthora (Desprez-Loustau et al., 2007). According to the COP 6 Decision VI/23 (2002, Hague, Netherlands), of the Convention on Biological Diversity (2002), the definition for invasive alien species is species, or lower taxon, which is present outside its natural distribution and whose introduction and/or spread threaten biological diversity. According to Brasier et al. (1999), the risk of emergence of new plant diseases is likely to increase as world trade in plants intensifies and more plants and their associated pathogens are introduced into new biogeographic environments. It raises the possibility closely related, but previously geographically isolated taxa, which lack strong reproductive barriers to come into contact. Many hybrids or introgressants are unlikely to be detected by the conventional methods, mainly morphologically based diagnostic methods used in international quarantine (Brasier et al., 1999). A brief description of some very dangerous Phytophthora species distributed in the world is following. Most of them are still not recorded in Bulgaria, but found in Europe. They are considered as potential very hazardous invaders for the forests and orchards in the country. Invasive Phytophthora pathogens with caduceus sporangia As already mentioned, some Phytophthora species have sporangia which are able to detach from the sporangiophores. They are known as species possessing caduceus sporangia, and undoubtedly, this trait gives them epidemiological advantage (Erwin and Ribeiro, 1996). Some of the most notorious Phytophthora spp., causing agents of severe diseases as P. infestans, P. capsici, P. ramorum, P. kernoviae and P. palmivora has caducous sporangia. From those P. infestans and P. capsici are largely distributed in Bulgaria on agricultural crops as potato, tomato and pepper. Since 1995 a new, never observed before disease on native California oak have been causing the death of tens of thousands trees along California and Oregon coast in USA. The disease was named Sudden Oak Death (SOD), or oak mortality syndrome. Since the time when the disease was first noted on tanoaks bordering a creek in California, until 1999 it turned into epidemic, resulting in tens of thousands dying native oaks (Garbelotto et al., 2001). The causing agent, a new Phytophthora sp. was isolated in 2000 and described in 2001 as Phytophthora ramorum sp. nov. This same pathogen has been detected several years earlier on ornamental plants across nurseries in Europe. For the first time it was found in 1993 on Rhododendron nursery stock in Germany and Netherlands. In 2003 nursery survey across European Union reveals more than 300 nurseries infected with P. ramorum only in UK, and infected nurseries in at least 11 other European countries. Soon after that, the pathogen was found spreading from nurseries to woodlands in Netherlands and UK, causing bleeding cankers and foliar lesions on Quercus, Fagus and other genera (Brasier and Jung, 2004). In 2010 P. ramorum continues to be the most intensively managed monitored and researched forest Phytophthora species in North America (Frankel and Hansen, 2011). Another invasive species, P. kernoviae was discovered when a range of surveys have been initiated for P. ramorum in the nurseries and in the woodlands of UK. The pathogen has overlapping, but smaller range of host plant species compare to P. ramorum. It is found to cause large bleeding cankers on European beech (Fagus sylvatica L.) and is serious foliar pathogen on Rhododendron spp. (Brasier et al., 2005). Phytophthora ramorum and Phytophthora kernoviae are both introduced, but the place of their origin is unknown. Both species have genetically uniform populations, which is explained with the bottleneck effect of introduction (Brasier, 2007). There are revealed four distinct evolutionary lineages of P. ramorum. Lineage NA1 is found in nurseries and forests in

4 Phytophthora spp. - Rising Threat for the Natural and Agro Ecosystems in Bulgaria 165 North America, lineage EU1 found both in Europe and North America. Third lineage NA2 was found only in nurseries in North America. The NA1 and NA2 isolates are both from mating type A2, as the isolates from EU 1 lineage are from A1 mating type (Grünwald et al., 2009). The fourth lineage EU2 recently was discovered on larch in Northern Ireland (Van Poucke et al., 2012). Both P. ramorum and P. kernoviae has caducous sporangia and air-born pattern of dissemination, they can cause symptomless sporulation on leaves of Rhododendron and other hosts, and they are able to colonize the xylem tissue (Brasier, 2007). Major difference between these two species is that P. ramorum is out crossing, as well as P. kernoviae is self-crossing (Webber, 2008). Another two new air-born species - Phytophthora nemorosa and Phytophthora folorium were discovered. P. nemorosa has remarkably similarity with P. ramorum in host range and symptomology and it was not possible to distinguish the diseases caused by these pathogens in the field. In contrast with P. ramorum, P. nemorosa is usually associated with single killed trees and has behavior suggesting an endemic pathogen (Hansen et al., 2003). The other species with caducous sporangia - Phytophthora foliorum is a pathogen on the azalea, causing leaf spot symptoms, and is not associated with significant mortality of azalea (Donahoo et al., 2006). Invasive Phytophthora pathogens with noncaduceus sporangia One of the most prominent cases of Phytophthora spp. with non-caduceus sporangia and soil-inhabiting lifestyle is P. cinnamomi. P. cinnamomi is a pathogen with a cosmopolite distribution and over 3000 host species. It is typical root pathogen and can grow saprophytically in the soil for long periods (Hardam, 2005 and Erwin and Ribeiro, 1996).The pathogen is responsible for the widespread damage in natural ecosystems in Western Australia. It is believed that the pathogen have been introduced in contaminated nursery stock in 1920 and spread into the jarrah (Eucalyptus marginata Donn ex Sm.) forests, probably from a centre of origin in the Papua New Guinea-Celebes region (Brasier, 1999) It is likely that the pathogen has a significant role in the mortality and decline of evergreen oaks in the beginning of the 1990s in the Mediterranean region (Brasier, 1999). P. cinnamomi has been widely spread also on conifers and woody ornamentals in nurseries and gardens across Europe during the 1960s and 70s. P. cinnamomi have also been associated with the little leaf disease of Pinus echinata in the 1930s. Symptomatic trees have been first evident adjacent to roads and forest tracks. The disease have been present across about one third of the tree s range, and have forced changes in management on about 2 million hectares of forest land. Both P. cinnamomi and P. cambivora are responsible for the devastating epidemic on the native European chestnut (Castanea sativa Miller) in the beginning of the 20 th century (Brasier, 1999). Another exotic pathogen Phytophthora lateralis, introduced probably with infected nursery stock from France in the far 1923 is posing a serious threat to cedar (Chamaecyparis lawsonia (Murray) Parl) forests in the Northwest and in British Columbia, Canada. The disease has been so severe in some localities, leading to the abandoning of the Lawson cypress plantings (Erwin and Ribeiro, 1996). The pathogen colonizes trees rapidly, advancing upward from infection points on fine roots until it reaches the main stem. Aerial infection can occur if the weather is humid, although P. lateralis has persistent sporangia (Hansen, 1999). A lethal Phytophthora disease threatens the alder population in Europe. It shows to be highly aggressive on alder trees (Alnus spp.), and is currently spreading in natural ecosystems across Europe (Ioos et al., 2006). It was observed first in southern United Kingdom in 1993 on Alnus glutinosa L., growing along rivers and in horticultural shelterbelts (Brasier et al., 2004; Streito and Gibbs, 1999). The majority of diseased trees are to be found on the banks of streams and rivers or on sites subject to flooding from adjacent watercourses. It has also been found in orchard shelterbelts and occasionally in new woodland plantings (Streito and Gibbs, 1999). Three years later the occurrence of the Alder disease was conformed from France. Tarry spots associated with a continuous basal lesion and crown transparency symptoms are highly characteristic of the disease (Streito and Gibbs, 1999). In Poland, trees with damaged roots did not have a cankerous discharge on trunks, just bark splitting (Oszako, 2007). Devastating alder Phytophthora appears to comprise a range of species hybrids. Brasier and colleagues describes it as a Phytophthora alni sp. nov. and its variants. The standard hybrid type is formally designated as Phytophthora alni subsp. alni (Brasier et al., 2004). It was shown to be near-tetraploid (Ioos et al., 2006), and the other unstable variant types, with chromosome numbers intermediate between diploid and tetraploid, were designated as P. alni subsp. uniformis; and P. alni subsp. multiformis. The results of pathogenicity experiments carried out over 4 year period ( ) shows that the isolates of the standard alder Phytophthora from several European countries are potentially highly aggressive pathogens of alder bark and appears to be relatively specific to alder. The other natural variants were comparatively weak pathogens, with one exception (Brasier and Kirk, 2001).

5 166 The standard alder Phytophthora type is widely distributed across Britain, Netherlands, France, Germany and Austria (Brasier, 2000). To the moment it is reported also from Belgium, Czech Republic, Hungary, Ireland, Italy, Lithuania, Poland, Slovakia, Slovenia, and Sweden ( Forestry Commission, Recently P. alni subsp. uniformis was reported to be isolated from soil beneath Alnus incana subsp. tenuifolia exhibiting dieback at two sample locations in remote, unmanaged stands hundreds of miles apart on the Kenai Peninsula and near Denali National Park in Alaska (Adams et al., 2008). Non caducous species causing aerial cankers The Phytophthora species which sporangia remain attached to the sporangiophores have soil inhabiting lifestyle and are predominantly root-infecting parasites (Cooke et al., 2000). In both southern England and Bavaria (Germany) it was found that the non-caducous soil and root inhabitants P. cambivora, P. citricola and P. gonapodyides can cause aerial bark lesions on beech (Jung et al., 2005). Also in UK P. citricola, which is a soil born species with non-caducous sporangia was found causing aerial bark lesions on European sycamore (Acer pseudoplatanus L.). It is still unknown how the inoculum of this soil borne pathogens becomes aerial. There were proposed two possible explanation for this unexpected pathogen behavior. One is the transport of inoculum via snails, which have been feeding on the exudates, and the other explanation might involve movement of inoculum via embolisms in infected xylem (Brasier and Jung, 2004). Interspecific hybrization within the genera Phytophthora In 1998 in The Netherlands from diseased ornamental plants (Spathiphyllum and Primula), grown on hydroponics system have been obtained three Phytophthora species, which have been proved to be interspecific hybrids between P. cactorum and P. nicotianae. Hydroponics systems facilitate physical encounters between different Phytophthora species, and it is possible natural hybridization processes to occur, as it is known that many different Phytophthora species are present in greenhouses. Since the introduction of hydroponic systems in greenhouses in the Netherlands, an increase of Phytophthora outbreaks has been recorded (Man in t Veld et al., 1998). Brasier and colleagues proposed that the standard alder Phytophthora is a recently evolved hybrid involving P. cambivora and a P. fragariae-like species as parents (Brasier et al., 2004; Ioos et al., 2006). In order to examine this hypothesis, Ioos and colleagues investigate the occurrence and the allelic distribution of four nuclear and two mitochondrial genes, on a wide collection of P. alni and closely related species. They favor the hypothesis that P. alni subsp. alni may have arisen via hybridization of two taxa close to and P. alni subsp. uniformis and P. alni subsp. multiformis, if not themselves. Sexual, rather than somatic, hybridization is more likely to have occurred. The authors guess that P. alni subsp. multiformis and P. alni subsp. uniformis, or P. alni subsp. multiformis-like and P. alni subsp. uniformis -like species, might have existed for a long time near alder trees before the recent emergence of large-scale decay in the European alder population. The occurrence of these species in the past might not have been noticed because of the lack of conspicuous symptoms or declines of whole trees (Ioos et al., 2006). In addition, the appearance of new hybrids in natural conditions, for instance P. alni and its subspecies is hardly to predict. The resulting new species might posses qualitatively new features-differences in virulence, host preferences and others. Climate change and Phytophthora Little is known about the influence of the climate change on the resident Phytophthora species, which appears not to be of great concern, as far as they do not cause significant diseases on their host plants. Much more attention is paid to the introduced species, and how their behavior will alter in answer of the global climate change. The second assessment report of the Intergovernmental Panel on Climate Change (IPCC), edited in 1995 concludes that the climate has changed over the past century in response to increasing atmospheric CO 2 with the global mean surface air temperature increasing between 0.3 and 0.6 C. Climate model simulations suggest an increase in global mean temperature between 1 and 3.5 C by the year The Kyoto Conference in December 1997 led to a climate treaty ratified by more than 150 countries. It is expected climate change to influence the geographical distribution and growth of plant species around the world. The most likely impacts of climate change would be shifts in the geographical distribution of host and pathogen, changes in the physiology of host-pathogen interactions, and changes in crop loss. Another important impact may be through changes in the efficacy of control strategies. The mechanism of pathogen dispersal, suitability of the environment for dispersal, survival between seasons, and any change in host physiology and ecology in the new environment will largely determine how quickly pathogens become estab-

6 Phytophthora spp. - Rising Threat for the Natural and Agro Ecosystems in Bulgaria 167 lished in a new region. Warming and other changes could also make plants more vulnerable to damage from pathogens that are currently not important because of unfavorable climate (Coakley et al., 1999). A series of computer models has been run to asses how predicted change in the mean annual temperature will alter the activity of P. cinnamomi in Europe. P. cinnamomi activity is constricted by both summer and winter temperatures. According to these models up to the year 2050, activity of P. cinnamomi is likely to increase significantly in the Mediterranean region and in maritime climates such as that of coastal western Britain, but not in central Europe (Brasier, 2000). There is a possibility the rise in the mean annual temperatures to lead to the increased activity of other forest Phytophthora spp. (Brasier, 2003). It is unknown what will be the influence of the possible climate changes on the endogenous species, and to what extend it might alter their ecological behavior. The problem with the worldwide spread of Phytophthora by human activities Movement of plants and plant products between biogeographically zones by human activities is now generally accepted to be the primary mode of introduction of exotic pathogens and pests (Brasier, 2008). There are many examples of introduced invasive species causing significant damage to both managed and natural ecosystems. The history of Phytophthora infestans appearance on potato crop in Ireland in 19 th century is the most frequently cited example for the consequences of introduction of an invasive species on new place. In a review from 1997, Goodwin summarizes the literature on the population genetic of Phytophthora available to that moment. He hypothesized that the first global migration of P. infestans has occurred on three stages. First it has appeared in the eastern United States in 1843, probably from Mexico on infected tubers. The second step in the migration took place during 1844 or 1845, probably in a single shipment of potatoes, and subsequently in almost all potato growing areas worldwide (Goodwin, 1997). Brasier (2007) outlines the problems behind the increased sudden appearance of pathogens as P. ramorum, P. kernoviae, P. alni and P. pinifolia. It is the growing threat to forests and natural ecosystems from invasive forest pathogens. The author is making a conclusion that although the international system is well regulated in many countries, for example North America and the U.K., it still has certain fundamental flaws. One is that many invasive pathogens were unknown to science until they escaped from their natural range, becoming an invasive. Unknown threats cannot be formally regulated against because they do not meet the requirement of being listable as a named threat organism. Phytophthora pathogen listed on quarantine schedules as causing damage on a specific host in particular country might be a serious threat to entirely different hosts and ecosystems in another country. The absence of that specific host is therefore insufficient reason to consider the pathogen non-threatening (Brasier, 2007). The appearance of P. lateralis on the wild growing Lawson cypress in the geologically and climatically distinctive Klamath ecological province in SW Oregon and NW California is another documentation of a system of unregulated international plant movements. The probable place of origin of P. lateralis might be Eastern Asia. It is supported by the tolerance of the Asian species to the pathogen (Hansen, 1999). Another issue is that visually healthy plants may harbour a sporulating pathogen in the roots or the foliage, and a visual inspection alone may be insecure. Thus shipping stock barerooted is not a guarantee of plant biosecurity, even more it might provide a false sense of security. The movement of inoculum on feet of people, animals and on wheeled vehicles is clearly a major potential pathway for local and even international spread of P. ramorum and P. kernoviae. At least two invasive Phytophthora species has been shown to spread on feet and machinery: P. cinnamomi in jarrah forests and native vegetation in Western Australia, and P. lateralis in Port-Orford-cedar areas in the Pacific Northwest (Brasier, 2007). Pest Risk Analysis (PRA) is the fundamental process, which is prescribed by the World Trade Organization Sanitary and Phytosanitary Agreement (WTO) as well as the International Plant Protection Convention (IPPC). The WTO Sanitary and Phytosanitary Agreement require that a scientific assessment of the risk posed by a plant pest is undertaken before any phytosanitary measures are set. Such measures must be the minimum needed to reduce the risk without unnecessarily impacting on trade. The organism in question must be identified as being a unique taxonomic entity. Recently, pest risk analysts have had to consider the risks posed by previously unidentified pathogens new to science (Sansford, 2009). Phytophthora spp. in Bulgaria In Bulgaria, species from genus Phytophthora are known generally as pathogens on some annual agricultural crops as potato, tomato, pepper and tobacco. They also cause problems in orchards and nurseries. Some Phytophthora species are causing rotting of ornamental plants grown in greenhouses (Stancheva and Rosnev, 2005). Phytophthora fragarie, a species that is included in the quarantine list is considered not to present in Bulgaria. In her DSc. thesis, Nakova (2012)

7 168 summarizes available information in the literature concerning the presence of Phytophthora species in the Bulgarian agro ecosystems, as well as in the semi-natural and natural ecosystems (Table 1). Nakova (2010) reported disease symptoms that are appearing on 2-3-year old apple trees in the region of Bjaga village (Plovdiv) during the period , and also on 2-year old cherry rootstocks (Katunitza, Plovdiv) and 2-year old cherries (Trilistnik, Plovdiv). She also is informing about Phythophthora infections of root and crown of cherries registered in Kjustendil, Sliven, Jambol, Karnobat, Bourgas and Svishtov regions, and on apples from Plovdiv (Bjaga, Asenovgrad and Tzalapitza), Kjustendil, Pazardzik and Haskovo (Stalevo) regions. She isolated and identified as Phytophthora cactorum, Phytophthora citrophthora and Phytophthora cryptogea, based on morphological and molecular traits from infected plant tissue from apple in Plovdiv and Kjustendil regions. She concludes that the disease spread was 2-3 % in most gardens, only in an apple orchard in Bjaga (Plovdiv region) it was up to 8-10 %. Phytophthora citricola and Phytophthora citrophthora have been reported from raspberry (Rubus idaeus L.) grown in a plantation of the experimental station of small fruits in Kostinbrod, Sofia region (Ilieva et al., 1995). All mentioned above Phytophthora species described as invasive and alien species are potentially extreme hazard plant pathogens for both natural and agro ecosystems in Bulgaria. Recently an international project for searching the biodiversity and distribution of Phytophthora species in the country and most of the Balkan region was conducted (Project ERA 138/01). It was considered as more detailed investigation of Phytophthora species in the natural ecosystems accomplished recently. In the frame of this project no one of the alien species discussed in this review, was found in the country. Nevertheless of this fact, larger, continues and more comprehensive monitoring is needed to be carried out regularly in order to cover all the specific and potentially inhabitance for the Phytophthora pathogens. Regulation of the movement of plants and plant products in Bulgaria In Bulgaria, the state policy in the field of pest control is carried out by the Ministry of Agriculture and Food through the National Plant Protection Service (NPPS), its regional offices for Plant Protection (RSPP) and the Central Laboratory for Plant Quarantine (CLPQ). National plant protection interacts with the competent authorities of other countries - EU members and the European Commission. The Minister of Agriculture and Food on the proposal of National Service approves the lists of pests, plants, plant products and other goods subject to phytosanitary control (LPP, 2011). Plant protection low and Ordinance 1 of 27 May 1998 on phytosanitary control is the legal basis of the state policy in plant protection. Only two species of Phytophthora are included in the quarantine lists: P. ramorum and P. fragariae, and are subject of phytosanitary control (LPP and Commission decision, 2002). If we compare this number with the number of known and the presumable number of unknown to science Phytophthora species it becomes clear that this is extremely insufficient. Intensifying the control and investigations of those plant pathogens is needed in the light of the discussed above impact of the climate change and human activities on the increased distribution and biodiversity of the Phytophthora spp. Methods for diagnostic of Phytophthora spp. Although Phytophthora spp. are aggressive pathogens, they are also seasonally active, delicate ephemeral organisms that are quickly replaced in host tissues by other fungi and by bacteria. It is therefore often difficult to isolate those pathogens for diagnosis, for taxonomic identification or for quarantine tests (Brasier, 1999). Some of the advantages and drawbacks of the available tools for detection and identification of Phytophthora species according to Cooke et al. (2007) are shown on Table 2. In regard to prevent introduction of new invasive pathogens in new geographical regions, which impact on the biodiversity in a given country is difficult to predict, it is important to remember the history of the appearance of newly emerged pathogens worldwide. We need to know the likely ways of the movement of the microorganisms between different points of the globe, and with the help of the continuously developing tools for detection, and more strict legislation rules in the world movement of goods to restrict the devastating impact of the human progress on the world biodiversity. The case with invasive Phytophthora pathogens is one of the many other cases of introduction of new organ- Law on the protection of plants. Reflected by the denomination on Prom. SG. 91 from 10 October 1997., amend. SG. issue 90 from 15 October 1999., amend. SG. issue 96 from 9 November 2001., suppl. SG. issue 18 from 5 March 2004., amend. SG. issue 26 from 28 March 2006., amend. SG. issue 30 from 11 April 2006., amend. issue SG. 31 from 14 April 2006., amend. SG. issue 96 from 28 November 2006., amend. SG. issue 13 from 8 February 2008., amend. SG. issue 36 of 4 April 2008., amend. SG. issue 43 of 29 April 2008., amend. SG. issue 82 from 16 October 2009., amend. SG. issue 8 from 25 January 2011., amend. SG. issue 28 from 5 April 2011(Bg). Ordinance 1 of 27 May 1998 on phytosanitary control. Effective from Issued by the Ministry of Agriculture, Forestry and Agrarian Reform. Prom. SG. issue 82 of 17 July 1998., amend. SG. issue 91 from 19 October 1999., amend. SG. issue 8 from 22 January 2002., amend. SG. issue 28 from 28 March, 2003., amend. SG. issue 7 from 24 January 2006., amend. SG. 75 from September 12, 2006., amend. SG. issue 82 from 12 October 2007., amend. SG. issue 82 from 19 September 2008., amend. SG. issue 8 from 30 January 2009., amend. SG. issue 30 from 21 April 2009., amend. SG. issue 98 from 11 December 2009.,amend. SG. issue 21 from 16 March 2010(Bg).

8 Phytophthora spp. - Rising Threat for the Natural and Agro Ecosystems in Bulgaria 169 Table 1 Phytophthora species reported in Bulgaria (according Nakova, 2012) Phytophthora spp. Hosts Authors Phytophthora parasitica var nicotianae Nicotiana tabacum Atanasov, 1928; Vanev et al., 1993 Lyccopersicon esculentum Vanev et al., 1993,; Ilieva 1979, 1990 Dianthus caryophyllus Vanev et al., 1993,; Ilieva 1979, 1990 Phytophthora parasitica var terestria Lyccopersicon esculentum Kovachevski and Hristov, 1929 Phytophthora infestans Solanum tuberosum Vanev et al., 1993 (The report is from Atanasov, 1934) Lyccopersicon esculentum Vanev et al., 1993 (The report is from Atanasov, 1934) Phytophthora erythroseptica Lyccopersicon esculentum Vanev et al., 1993 (The report is from Atanasov and Kovachevski, 1929) Solanum tuberosum Vanev et al., 1993 (The report is from Atanasov and Kovachevski, 1929) Phytophthora erythroseptica var. erythroseptica Lyccopersicon esculentum Ilieva, 1990 Phytophthora cryptogea var. richardiae Calla aetiopica Hristova, 1941 Phytophthora cryptogea Phytophthora cactorum (Lebert et Cohn) Phytophthora capsici (Leonian) Lyccopersicon esculentum Ilieva 1979, 1990 Capsicum annum Ilieva, 1990; Ilieva et al., 1992 Fragaria spp. Ilieva, 1990; Ilieva et al., 1992 Cucumis melo Ilieva, 1990; Ilieva et al., 1992 Nicotiana tabacum Ilieva, 1990 Leontopodium alpinum Ilieva, 1990; Ilieva et al., 1992 Beta vulgaris Ilieva, 1990; Ilieva et al., 1992 Calceolaria sp. Vanev et al., 1993 Clarkia sp. Vanev et al., 1993 Fragaria elatior Vanev et al., 1993 Paeonia pelegrina Vanev et al., 1993 Panax quinquefolius Bobev, 2003 Fragaria sp. Ilieva, 1995 Capsicum annum Kovachevski, 1934; Elenkov and Bahriev, 1975; Elenkov and Hrelkova, 1977; Elenkov and Hristova, 1978; Ilieva, 1976, 1979, 1990; Vanev et. al, 1993; Georgieva, 1985 Citrulus vulgaris Vanev et al., 1993 Dianthus caryophyllus Ilieva, 1988;Vanev et al., 1993; Lycopesicum esculentum Vanev et al., 1993, Vanev et al., 1993 Melo sativus Ilieva, 1990; Vanev et al., 1993 Solanum melongena Ilieva, 1990; Vanev et al., 1993 Fragaria elatior Vanev et al., 1993 Phytophthora citricola Fragaria sp. Ilieva, 1995 Rubus sp. Ilieva, 1995 Phytophthora dreschleri Cucumis sativus Ilieva, 1985, 1990;Vanev et al., 1993 Cucumis melo Ilieva, 1985, 1990 Phytophthora fragariae Fragaria spp. Vanev et al., 1993; Nakov, 1976 Phytophthora richardiae Zantedeschia aetiopica Vanev et al., 1993 Phytophthora megasperma Fragaria sp. Ilieva, 1995

9 170 Table 2 Tools for detection and identification of Phytophthora species according to Cooke et al. (2007) Traditional detection methods Approach Advantages Drawbacks Visual detection for signs Limited to restricted number of host-pathogen combinations in of Phytophthora on infected which airborne spores are visible as a downy mass of sporulation plant parts and direct (Cooke et al., 2007) In woody trees symptoms might not be microscopic examination of detected even if the tree have lost 50% of its lateral roots (Erwin diseased material and Ribeiro, 1996). Baiting with plant material and Isolation of the pathogen from infected plant tissues, water and soil, using general or selective agar media Use of antibodies (molecules produced by the mammalian immune systems to identify invading organisms or substances. Common and appropriate approach, cost effective, simple and familiar to many workers. In the case of river sampling the bait can be left in the substrate for relatively long periods and may thus recover low levels of Phytophthora. Serological methods Monoclonal antibodies have been utilized for the production of Phytophthora species and genus specific commercial detection kits, using both membrane-based and ELISA formats. Some of these kits proved appropriate for the identification of important forest Phytophthoras. Molecular methods The number of species detected might be strongly reduced due to the selective colonization and development on different types of baits. The oospore population might be not detected. Masking or inhibition of the relatively slowly growing Phytophthora by other competitive microorganisms. Assessments based solely on baiting are likely to underestimate the diversity. Monoclonal antibodies are generally slow to produce, expensive to both produce and maintain, and occasionally cell lines may die or stop producing the required antibody. Conventional PCR The most important and sensitive technique presently available for the detection of plant pathogens. Microorganisms do not need to be cultured. Single target molecule can be detected in a complex mixture The adoption of PCR for routine detection of plant pathogens is relatively slow, often due to technical limitations related to the post-amplification amplicon detection procedures. Nested PCR Real-time PCR Multiplex real time PCR Improved sensitivity and specificity compared to conventional PCR Provides a more rapid means for screening water, plant and soil samples. Eliminates the requirement for post amplification processing steps thus saving time and labor, compared to conventional PCR. Reduces the risk of cross contamination. It is suitable for estimating plant pathogen biomass in host tissues or environmental samples. It is not affected by external factors such as other fungal species that could conceal the presence of the pathogen. Enable detection multiple templates within a single reaction, particularly important for Phytophthora species in forest ecosystems, where they are frequently found in clusters on the same site(tree) Increased sensitivity in comparison to other detection methods, including conventional PCR. Increases the risk of false positives due to cross contamination and involves more time and effort. DNA micro-and macroarrays Enables the simultaneous detection of many plant pathogens. Macro-arrays have proved appropriate for the multiplex detection of plant pathogens from complex environmental samples including those derived from soil and plants. Direct sequencing from environmental samples Lack of cross-reaction with the ubiquitous Pythium species. Enables the detection of many Phytophthora species.

10 Phytophthora spp. - Rising Threat for the Natural and Agro Ecosystems in Bulgaria 171 isms from all existing taxa, which turns into causing agents of devastating epidemics. Conclusion In regard to prevent introduction of new invasive pathogens in new geographical regions, which impact on the biodiversity in a given country is difficult to predict, it is important to remember the history of the appearance of newly emerged pathogens worldwide. We need to know the likely ways of the movement of the microorganisms between different points of the globe, and with the help of the continuously developing tools for detection, and more strict legislation rules in the world movement of goods to restrict the devastating impact of the human progress on the world biodiversity. The case with invasive Phytophthora pathogens is one of the many other cases of introduction of new organisms from all existing taxa, which turns into causing agents of devastating epidemics. 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